Coccidioides spp. are fungal pathogens that normally causes a pneumonia associated with considerable morbidity and attendant economic or medical care costs. Some infections produce respiratory failure, soft tissue abscesses, osteomyelitis or meningitis. Even in otherwise healthy persons, inhalation of a single spore is sufficient to result in death from one or more of these complications, and this degree of infectivity was responsible, in part, for past development programs by the U.S. and the Soviet Union to use Coccidioides spp. as biological weapons. Coccidioides spp., recently classified as Category C agents by NIAID, are also considered emerging public health pathogens. Because coccidioidal infection so often produces life-long protection against reinfection, it is likely that a preventative vaccine could be effective. A screening program of approximately two dozen coccidioidal proteins identified a recombinant vaccine that was recommended for clinical trials. However, manufacturing difficulties related to the antigen's specific sequence has impeded its further development. In this project, we shall use in vitro protein expression to permit antigen screening on a larger scale as a means of identifying equally protective antigens that are more amenable to formulation. A proteomic analysis of spherule cell walls has identified 650 proteins and of these we shall screen approximately 1,000 exons with the least similarity to mammalian proteins, using first seroreactivity and subsequently reactivity of lymphocytes from mice previously infected with Coccidioides or immunized with protective whole cell vaccines. Our goal is to identify a second-generation recombinant vaccine candidate that could be developed for clinical testing. This project will also use tandem mass spectrometry to develop antigen-detecting assays as a more sensitive diagnostic for early Valley Fever. We have recently detected in the lungs of infected mice dozens of coccidioidal proteins. Three of these have no similarity to mammalian proteins and <45% similarity to other fungal proteins. We shall express these biosignature targets by recombinant methods and use the purified proteins to raise high-affinity antibodies. Tandem mass spectrometry will also be used to characterize the strength and specificity of antigen-antibody interactions to serve as independent analyses for the rational and optimal design of direct fluorescent staining or ELISA methods to detect antigens in clinical specimens.